Plant pathogens cause hundreds of millions of dollars in damage to crops in the United States annually and cause significantly more damage worldwide. Traditional plant breeding techniques have developed some plants that resist specific pathogens, but these techniques are limited to genetic transfer within breeding species and can be plagued with the difficulty of introducing non-agronomic traits that are linked to pathogen resistance. Furthermore, traditional breeding has focused on resistance to specific pathogens rather than general, or systemic, resistance to a wide spectrum of pathogens. Therefore, an important goal in agriculture is to identify genetic components that enable plants to resist pathogens, thereby allowing for the development of systemically resistant plants through biotechnology.
Systemic acquired resistance (SAR) is a general plant resistance response that can be induced during a local infection by an avirulent pathogen. While early studies of SAR were conducted using tobacco mosaic virus (TMV) and its Solanaceous hosts (see, e.g., Ross, A. F. Virology 14: 340-358 (1961)), SAR has been demonstrated in many plant species and shown to be effective against not only viruses, but also bacterial and fungal pathogens (see, e.g., Kuc, J. Bioscience 32:854-860 (1982) and Ryals, et al., Plant Cell 8:1809-1819 (1996)). A necessary signal for SAR induction is salicylic acid (SA); plants that fail to accumulate SA due to the expression of an SA-oxidizing enzyme salicylate hydroxylase are impaired in SAR (Gaffney, T., et al. Science 261:754-756 (1993)). Conversely, an elevation in the endogenous level of SA or exogenous application of SA or its synthetic analogs, such as 2,6-dichloroisonicotinic acid (INA), not only results in an enhanced, broad-spectrum resistance but also stimulates concerted expression of a battery of genes known as pathogenesis-related (PR) genes (see, e.g., Malamy, J., et al. Science 250:1002-1004 (1990); Metraux, J.-P., et al. Science 250:1004-1006 (1990); Rasmussen, J. B., et al. Plant Physiol 97:1342-1347 (1991); Yalpani, N., et al. Plant Cell 3:809-818 (1991); White, R. F. Virology 99:410-412 (1979); Metraux, J.-P., et al. (1991) In Advances in Molecular Genetics of Plant-Microbe Interactions, eds. Hennecke, H. & Verma, D. P. S. (Kluwer Academic, Dordrechet, The Netherlands), Vol. 1, pp. 432-439; Ward et al. Plant Cell 3:1085-1094 (1991); and Uknes et al. Plant Cell 4:645-656 (1992)). PR genes may play direct roles in conferring resistance because their expression coincides with the onset of SAR and some of the PR genes encode enzymes with antimicrobial activities (see, e.g., Ward et al. Plant Cell 3:1085-1094 (1991); and Uknes et al. Plant Cell 4:645-656 (1992)). Therefore, understanding the regulation of PR gene expression has been a focal point of research in plant disease resistance.
Two classes of A. thaliana mutants with altered PR gene expression have been identified. One class constitutively expresses PR genes while the other class is impaired in the SA- or INA-induced PR gene expression (Lawton, K., et al. (1993) in Mechanisms of Defense Responses in Plants, eds. Fritig, B. & Legrand, M. (Kluwer Academic, Dordrecht, The Netherlands), pp. 422-432; Bowling, S. A., et al. Plant Cell 6:1845-1857 (1994); Bowling, S. A., et al. Plant Cell 9:1573-1584 (1997); Clarke, J. D., et al. Plant Cell 10:57-569 (1998); Cao, H., et al. Plant Cell 6:583-1592 (1994); Delaney, T. P., et al. Proc. Natl. Acad. Sci. USA 92:602-6606 (1995); Glazebrook, J., et al., Genetics 143, 973-982 (1996); Shah, J., et al. Mol. Plant-Microbe. Interact. 10:69-78 (1997)). Interestingly, from the second class of mutants only one genetic locus, NPR1 (also known as NIMI), has been identified. NPR1 has been shown to be a key component of the SA-regulated PR gene expression and disease resistance because nprl mutants fail to express PR1, PR2, and PR5 and display enhanced susceptibility to infection even after treatment with SA or INA. Furthermore, transgenic plants overexpressing NPR1 display a more dramatic induction of PR genes during an infection and show complete resistance to Pseudomonas syringae pv. maculicola 4326 and Peronospora parasitica Noco, two very different pathogens that are virulent on wild-type A. thaliana plants (Cao, H., et al. Proc. Natl. Acad. Sci. USA 95:6531-6536 (1998)).
Sequence analysis of NPR1 does not reveal any obvious homology to known transcription factors (see, e.g., Cao, H., et al. Cell 88:57-63 (1997) and Ryals, J. A., et al. Plant Cell 9:425-439 (1997)). Therefore, it is unlikely that NPR1 is directly involved in transactivating the promoters of PR genes. However, NPR1 contains at least four ankyrin repeats, which are found in proteins with very diverse biological functions and are involved in protein-protein interactions (Bork, P. (1993) Proteins: Structure, Function, and Genetics 17, 363-374. Michaely, P., and Bennet, V. (1992) Trends in Cell Biology 2:127-129.). The functional importance of the ankyrin repeat domain has been demonstrated by mutations found in the npr1-1 and the nim1-2 alleles where the highly conserved histidine residues in the third and the second ankyrn repeats, respectively, are changed to a tyrosine. Because these conserved histidine residues are involved in the formation of hydrogen bonds which are crucial in stabilizing the three dimensional structure of the ankyrin-repeat domain (Gorina, S. & Pavletich, N. P. Science 274, 1001-1005 (1996)), npr1-1 and nim1-2 mutations may cause disruption in the local structure within the ankyrin-repeat domain and abolish its ability to interact with other proteins. These data suggest that NPR1 probably exerts its regulatory function by interacting with other proteins.
SA-responsive promoter elements such as the as-1 element in the 35S promoter of cauliflower mosaic virus (CaMV) and the ocs and nos elements in opine synthase promoters of Agrobacterium have previously been identified and characterized (Lam, E., et al. Proc. Natl. Acad. Sci. USA 86, 7890-7894 (1989); Qin, X-F., et al. Plant Cell 6, 863-874 (1994) ; and Ellis, J. G., et al. Plant J. 4, 433-443 (1993)). The as-1 element has been shown to bind to a tobacco transcription factor, SARP (salicylic acid response protein), which is immunologically related to the tobacco protein TGA1a, a bZIP transcription factor (Jupin, I. & Chua N-H. (1996) EMBO J. 15:5679-5689). In A. thaliana, there are at least six bZIP genes identified that have homology to the tobacco TGA transcription factor (Kawata, T., et al. Nucleic Acids Res. 20, 1141 (1992); Xiang, C., et al. Plant Mol. Biol. 34, 403-415 (1997); Zhang, B., et al. Plant J. 4,711-716 (1993 ); Schindler, U., et al., A. R. Plant Cell 4, 1309-1319 (1992); Miao, Z. H., et al. Plant Mol. Biol. 25, 1-11 (1994); and Lam, E. & Lam, Y. K.-P. Nucleic Acids Res. 23, 3778-3785 (1995)). These TAG transcription factors have been shown to have different affinities for the as-1 element in in vitro binding assays (Lam, E. & Lam, Y. K.-P. Nucleic Acids Res. 23, 3778-3785 (1995)). While strong, binding of AHBP-1b requires two tandem copies of the TGACG motif present in the as-1 element, binding of TGA6 appears to be unaffected by the number of motifs because a single copy seems to be sufficient. Other bZIP genes have been identified in wheat (see, e.g., Foley et al., Plant J. 3(5):669-79 (1993) and tobacco (see, e.g., Fromm, et al, Mol. Gen. Genet. 229:181-88 (1991) and Katagiri et al., Nature 340:727-30 (1989)). Although functions have been postulated for some of the above-described bZIP gene products, little is known about the regulation of bZIP gene products and there are no reports of their interaction with any other proteins associated with plant disease resistance.
Recently, the promoter of the A. thaliana PR-1 gene has been thoroughly analyzed using deletion and linker scanning mutagenesis performed in transgenic plants as well as in vivo footprinting analysis (Lebel, E., et al. Plant J. 16, 223-234 (1998)). Through these analyses, two INA-responsive elements have been defined. One element at -610 is similar to a recognition sequence for the transcription factor NF-.kappa.B, while the other promoter element around residue -640 contains a CGTCA motif (the complementary sequence is TGACG) which is present in the as-1 element. The CGTCA motif was shown by linker-scanning mutagenesis to be essential for both SA and INA induction of PR-1 gene expression.
In spite of the recent progress in understanding the genetic control of plant resistance to pathogens, little progress has been reported in the identification and analysis of genes interacting with key regulators of pathogen resistance such as NPR1. Characterization of such genes would allow for the genetic engineering of plants with a variety of desirable traits. The present invention addresses these and other needs.